The effect of autophagy on hemoporfin-mediated photodynamic therapy in human umbilical vein endothelial cells.

SIGNIFICANCE: Hemoporfin-mediated photodynamic therapy (HMME-PDT) has been recognized as a safe and effective treatment for port wine stain (PWS). However, some patients show limited improvement even after multiple treatments. Herein, we aim to explore the effect of autophagy on HMME-PDT in human umbilical vein endothelial cells (HUVECs), so as to provide theoretical basis and treatment strategies to enhance clinical effectiveness. METHODS: Establish the in vitro HMME-PDT system by HUVECs. Apoptosis and necrosis were identified by Annexin Ⅴ-FITC/PI flow cytometry, and autophagy flux was detected by monitoring RFP-GFP-LC3 under the fluorescence microscope. Hydroxychloroquine and rapamycin were employed in the mechanism study. Specifically, the certain genes and proteins were qualified by qPCR and Western Blot, respectively. The cytotoxicity was measured by CCK-8, VEGF-A secretion was determined by ELISA, and the tube formation of HUVECs was observed by angiogenesis assay. RESULTS: In vitro experiments revealed that autophagy and apoptosis coexisted in HUVECs treated by HMME-PDT. Apoptosis was dominant in early stage, while autophagy gradually increased in the middle and late stage. AMPK, AKT and mTOR participated in the regulation of autophagy induced by HMME-PDT, in which AMPK was positive regulation, while AKT and mTOR were negative regulation. Hydroxychloroquine could not inhibit HMME-PDT-induced autophagy, but capable of blocking the fusion of autophagosomes with lysosome. Rapamycin might cooperate with HMME-PDT to enhance autophagy in HUVECs, leading to increased cytotoxicity, reduced VEGF-A secretion, and weakened angiogenesis ability. CONCLUSIONS: Both autophagy and apoptosis contribute to HMME-PDT-induced HUVECs death. Pretreatment of HUVECs with rapamycin to induce autophagy might enhance the photodynamic killing effect of HMME-PDT on HUVECs. The combination of Rapamycin and HMME-PDT is expected to further improve the clinical efficacy.

Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood.

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.

Evaluation of HPV infection helps to direct ALA-PDT of condyloma acuminata.

BACKGROUND AND OBJECTIVES: Aminolaevulinic acid-mediated photodynamic therapy (ALA-PDT) is increasingly applied for the treatment of condyloma acuminata (CA). However, the determinants for the sessions and end points of ALA-PDT treatment remains unclear. Here, we recorded HPV screening, evaluated the frequency and efficacy of ALA-PDT in different types of CA, in order to individualize ALA-PDT treatment to CA. METHODS: A total of 227 CA patients with HPV infection and visible warts were recruited. Prior to PDT, visible lesions were removed by radio frequency or microwave. HPV DNA detection were performed before each PDT treatment and at follow-up. Treatment was ended after two consecutive negative HPV DNA detection. RESULTS: Of the 227 patients, 119 patients received ALA-PDT and 116 patients completed all treatments. We found that CA patients with multiple-site infection, intra-luminal infection or multiple-type of HPV infection required more sessions of ALA-PDT. The recurrence rate was 8.62% (10/116). Viral load was significantly lower after six PDT treatments compared to viral load after three PDT treatments. Gender, HPV subtypes and warts location had no significant effect on the recurrence rate. CONCLUSION: Comprehensive evaluation of HPV infection state helps to individualize ALA-PDT treatment scheme for CA patients and predict the therapeutic efficacy.

ERK/MEK Pathway Regulates Th17 Cell Differentiation in Patients with Pemphigus Vulgaris.

Background: T helper (Th) cells are involved in the pathogenesis of pemphigus vulgaris (PV). However, the mechanism still needs more exploration. Aims: This study aimed to evaluate the molecular mechanism of the dysregulation of Th17 cells in the peripheral blood of patients with PV. Materials and Methods: Serum levels of IL-17 and anti-Dsg3 titres in patients with PV were analysed using ELISA. The mRNA expression of retinoic acid orphan receptor γt (RORγt) in CD4+ T cells was detected using reverse transcription-quantitative PCR (qPCR). The number of Th17 cells was examined using flow cytometry. Western blot analysis and immunofluorescent staining were also performed to investigate the expression levels of ERK/MAPK signalling proteins and Th17 lineage-associated proteins. Results: The proportion of Th17 cells and Th17 spectrum-associated proteins (p-STAT3, RORγt and IL-17) were upregulated in CD4+ cells in PV patients. The increased transcriptional levels of RORγt and IL-17 correlated positively with the severity of PV. Elevated phosphorylation of the ERK signalling factors was found in the collected CD4+ T cells in PV patients. The inhibition of the ERK signalling pathway significantly reduced the differentiation of Th17 cells in PV patients in vitro. Conclusion: Th17 cells are essential in the dysregulation of PV, and ERK signalling is involved in Th17-type immunity and promotes the development of PV. The study here provides us with a potential therapeutic target for PV.

Podophyllotoxin-combined 5-aminolevulinic acid photodynamic therapy significantly promotes HR-HPV-infected cell death.

BACKGROUND: Human papillomavirus (HPV) infection and related diseases are difficult clinical challenges. The efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in treating condyloma acuminata is remarkable, with high virus clearance and low recurrence rates. Podophyllotoxin (POD) is the first-line drug with a significant therapeutic effect on condyloma acuminata. However, no studies have determined whether POD-combined ALA-PDT improves high-risk (HR)-HPV-infected cell killing. We aimed to investigate whether POD-combined ALA-PDT could promote HPV-infected cell death more effectively than the single treatment and explore the underlying mechanism. METHODS: In HeLa and SiHa cells, flow cytometry, EdU assay and LDH release test were used to detect apoptosis, cell proliferation change and necrosis, respectively. To investigate whether the combined therapy might activate apoptosis and induce endoplasmic reticulum (ER) stress, flow cytometry was used to determine intracellular levels of ROS and calcium, and Western blotting was used to determine the expression of related proteins. Mitochondrial membrane depolarization was detected by JC-1 assay. Immunofluorescence staining and Western blotting were used to detect the activation of autophagy. RESULTS: Podophyllotoxin -combined ALA-PDT inhibited the proliferation and promoted apoptosis and necrosis more effectively than the single treatment at the same intensity and concentration. The activation of the caspase-dependent apoptosis pathway, ER stress and autophagy was more substantial in POD-combined ALA-PDT than with single treatments. CONCLUSION: Podophyllotoxin -combined ALA-PDT effectively promoted cell death through several pathways in HeLa and SiHa cells. This combination might be a promising therapeutic strategy for the HR-HPV infection.

Zinc Oxide Nanoparticles Prime a Protective Immune Response in Galleria mellonella to Defend Against Candida albicans.

Purpose: Zinc oxide nanoparticles (ZnO-NPs) have exerted antimicrobial properties. However, there is insufficient evaluation regarding the in vivo antifungal activity of ZnO-NPs. This study aimed to investigate the efficacy and mechanism of ZnO-NPs in controlling Candida albicans in the invertebrate Galleria mellonella. Methods: Galleria mellonella larvae were injected with different doses of ZnO-NPs to determine their in vivo toxicity. Non-toxic doses of ZnO-NPs were chosen for prophylactic injection in G. mellonella followed by C. albicans infection. Then the direct in vitro antifungal effect of ZnO-NPs against C. albicans was evaluated. In addition, the mode of action of ZnO-NPs was assessed in larvae through different assays: quantification of hemocyte density, morphology observation of hemocytes, characterization of hemocyte aggregation and phagocytosis, and measurement of hemolymph phenoloxidase (PO) activity. Results: Zinc oxide nanoparticles were non-toxic to the larvae at relatively low concentrations (≤20 mg/kg). ZnO-NP pretreatment significantly prolonged the survival of C. albicans-infected larvae and decreased the fungal dissemination and burden in the C. albicans-infected larvae. This observation was more related to the activation of host defense rather than their fungicidal capacities. Specifically, ZnO-NP treatment increased hemocyte density, promoted hemocyte aggregation, enhanced hemocyte phagocytosis, and activated PO activity in larvae. Conclusion: Prophylactic treatment with lower concentrations of ZnO-NPs protects G. mellonella from C. albicans infection. The innate immune response primed by ZnO-NPs may be part of the reason for the protective effects. This study provides new evidence of the capacity of ZnO-NPs in enhancing host immunity and predicts that ZnO-NPs will be attractive for further anti-infection applications.

Case Report: Short-Term Application of Topical Imiquimod Is Practical for Chromoblastomycosis.

Chromoblastomycosis is a chronic cutaneous fungal infection caused by dematiaceous fungi. It is a therapeutic challenge because of the lack of specific treatments. We describe a refractory case of chromoblastomycosis in which the lesion did not respond to initial treatment, but then use of topical imiquimod cured the lesion successfully.

mTOR pathway regulates the differentiation of peripheral blood Th2/Treg cell subsets in patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease. Aberrant mTOR pathway activity is involved in many autoimmune diseases. This study investigated the correlation of mTOR pathway (PI3K/AKT/mTOR/p70S6K) activity with the loss of balance in T helper 2/regulatory T (Th2/Treg) cells in the peripheral blood of PV patients. CD4+ T cells were isolated from 15 PV patients and 15 healthy controls (HCs), the ratios of Th2/CD4+ T cells and Treg/CD4+ T cells, the activity of the mTOR pathway (PI3K/AKT/mTOR/p70S6K), the transcription factors and cytokines of Th2 and Treg cells were detected. Primary CD4+ T cells from PV patients were cultured under Th2- or Treg-polarizing conditions with or without rapamycin in vitro. We found that PV patients showed significantly elevated serum IL-4 when compared with HCs, and serum IL-4 level was positively correlated with the titer of anti-Dsg1/3 antibody and disease severity, while the serum TGF-ß level was negatively correlated with the titer of anti-Dsg3 antibody and disease severity. Meanwhile, PV patients showed increased Th2/CD4+ T cell ratio; decreased Treg/CD4+ T cell ratio; elevated mRNA of PI3K, AKT, mTOR and protein of PI3K (P85), AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), GATA3; reduced protein of forkhead box protein 3. Rapamycin inhibited Th2 cell differentiation and promoted Treg cell differentiation in vitro. These data suggest a close association between mTOR pathway activation and the loss of balance in Th2/Treg cells in peripheral blood of PV patients. Inhibiting mTORC1 can help restore the Th2/Treg balance.

Rare cases of PAMI syndrome in both father and son with the same missense mutation in PSTPIP1 gene and literature review.

PSTPIP1-associated myeloid-related proteinaemia inflammatory (PAMI) syndrome has been described as a rare and distinct clinical phenotype of PSTPIP1-associated inflammatory diseases. We report PSTPIP1 mutation in both father and son who have leukopenia and acne-like lesions. Through whole-exome sequencing on blood DNA, it is found a heterozygous mutation of PSTPIP1 gene c.748G>A on the father and son. The diagnosis of PAMI is made based on DNA sequencing results and clinical characteristics of typical lesions, leukopenia, and the markedly increased serum S100A8/A9 (calprotectin).

Pre-exposure to Candida glabrata protects Galleria mellonella against subsequent lethal fungal infections.

Commensal fungi are an important part of human microbial community, among which Candida albicans and Candida glabrata are two common opportunistic pathogens. Unlike the high pathogenicity of C. albicans, C. glabrata is reported to show low pathogenicity to the host. Here, by using a Galleria mellonella infection model, we were able to confirm the much lower virulence of C. glabrata than C. albicans. Interestingly, pre-exposure to live C. glabrata (LCG) protects the larvae against subsequent various lethal fungal infections, including C. albicans, Candida tropicalis, and Cryptococcus neoformans. Inconsistently, heat-inactivated C. glabrata (HICG) pre-exposure can only protect against C. albicans or C. tropicalis re-infection, but not C. neoformans. Mechanistically, LCG or HICG pre-exposure enhanced the fungicidal activity of hemocytes against C. albicans or C. tropicalis. Meanwhile, LCG pre-exposure enhanced the humoral immunity by modulating the expression of fungal defending proteins in the cell-free hemolymph, which may contribute to the protection against C. neoformans. Together, this study suggests the important role of C. glabrata in enhancing host immunity, and demonstrates the great potential of G. mellonella model in studying the innate immune responses against infections.

5-aminolevulinic acid photodynamic therapy and excision surgery for nevoid basal cell carcinoma syndrome with multiple basal cell carcinomas and PTCH1 mutation.

This report describes a PTCH1 c.1804C > T (p.Arg602*) mutation causing a Chinese nevoid basal cell carcinoma syndrome (NBCCS) with multiple basal cell carcinoma (BCC) phenotype. Multiple modalities including microwave ablation, photodynamic therapy, and excision surgery have a good respond to the NBCCS. The current results broaden the spectrum of PTCH1 mutations responsible for NBCCS.

Antimicrobial and immunomodulatory responses of photodynamic therapy in Galleria mellonella model.

BACKGROUND: New therapeutics are urgently needed for infectious diseases, especially for the fungal infection like Fonsecaea monophora. Photodynamic therapy has been showing antimicrobial activity on some pathogens. The combination of antimicrobial medicines and photodynamic therapy (PDT) might be a practical approach. However, whether the treatment of PDT could do benefits to the host immunity remains poorly documented. RESULTS: In this study, Galleria mellonella larvae were employed as a model organism to evaluate the activity of PDT, and also to investigate the regulation of humoral immunity by PDT. Photosensitizer 5-aminolevulinic acid (ALA) was applied to the G. mellonella infection model. It was found that ALA-mediated PDT was non-toxic to G. mellonella, and could extend the median survival of infected larvae from 3 days to 5.5 days. We observed that larval hemocytes inhibited the growth of Candida albicans and Staphylococcus aureus, without any contribution by ALA-PDT. Furthermore, the application of ALA-PDT demonstrated the immunomodulation of larval innate immunity as increased hemocyte density counting, cell morphological transformation, and sensitivity to pathogens. CONCLUSIONS: G. mellonella could be considered as a useful model to study the immunoregulation of PDT. This model revealed that ALA-PDT positively defense against infections through inducing humoral immune responses of larvae.

Increased expression of miR-338-3p impairs Treg-mediated immunosuppression in pemphigus vulgaris by targeting RUNX1.

Pemphigus vulgaris (PV) is a regulatory T cell (Treg)-associated autoimmune disease. Treg cells maintain immunosuppression by expressing the signature transcription factor FOXP3. MicroRNAs (miRNAs) have frequently emerged as regulators in Treg-mediated immunosuppression. We previously found that miR-338-3p was overexpressed in the peripheral blood mononuclear cells of patients with PV. Herein, we explored the role of miR-338-3p in Treg-mediated immunosuppression by quantitative real-time polymerase chain reaction, analysis of public microarray data, miRNA transfection, Western blotting, flow cytometry, and luciferase reporter assays. Increased expression of miR-338-3p was detected in CD4+ T cells of active PV patients compared with those in healthy controls. Moreover, the miR-338-3p level was positively related to disease severity. Bioinformatics prediction revealed that Runt-related transcription factor 1 (RUNX1), a gene activating FOXP3 expression, was a putative target of miR-338-3p. There was a reduction of FOXP3 and RUNX1 expression in the CD4+ T cells of patients with PV, along with significant correlations with the level of miR-338-3p. MiRNA transfection, mRNA and protein analysis, and luciferase reporter assays verified that miR-338-3p attenuated FOXP3 expression by targeting RUNX1. This study suggests that excessive expression of miR-338-3p attenuates the expression of FOXP3 by targeting RUNX1, contributing to Treg dysfunction in PV.

Exome sequencing identifies a KRT9 pathogenic variant in a Chinese pedigree with epidermolytic palmoplantar keratoderma.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is a rare skin disorder and its pathogenesis and inheritability are unknown. OBJECTIVE: To investigate the inheritance and pathogenesis of EPPK. METHODS: Two EPPK cases occurred in a three-generation Chinese family. Patient-parents trio EPPK was carried out and the identified candidate variants were confirmed by Sanger sequencing. RESULTS: A heterozygous missense pathogenic variant, c.488G > A (p.Arg163Gln), in the keratin (KRT) 9 gene was detected in the proband and his son via targeted exome sequencing, and then validated by Sanger sequencing. This pathogenic variant cosegregated with the EPPK in extended family members, and was predicted to be pathogenic by SIFT, PolyPhen2, PROVEAN, and Mutation Taster. This heterozygous variation was not evident in 100 healthy controls. CONCLUSION: This report describes a KRT9 c.488G > A (p.Arg163Gln) variant causing a diffuse phenotype of Chinese EPPK. The current results broaden the spectrum of KRT9 pathogenic variants responsible for EPPK and have important implications for molecular diagnosis, treatment, and genetic counseling for this family.

n-3 polyunsaturated fatty acids abrogate mTORC1/2 signaling and inhibit adrenocortical carcinoma growth in vitro and in vivo.

n-3 polyunsaturated fatty acids (PUFAs) are essential for human health and have been reported to reduce the risk of cancer, inhibit the growth of various types of tumors both in vitro and in vivo, and affect adrenal function. However, their effects on adrenocortical carcinoma (ACC) are not known. In the present study, we demonstrated that docosahexenoic acid (DHA) inhibited ACC cell proliferation, colony formation and cell cycle progression, and promoted apoptosis. In addition, ectopic expression of fat-1, a desaturase that converts n-6 to n-3 PUFAs endogenously, also inhibited ACC cell proliferation. Moreover, supplementing n-3 PUFAs in the diet efficiently prevented ACC cell growth in xenograft models. Notably, implanted ACC cells were unable to grow in fat-1 transgenic severe combined immune deficiency mice. Further study revealed that exogenous and endogenous n-3 PUFAs efficiently suppressed both mTOR complex 1 (mTORC1) and mTORC2 signaling in ACC in vitro and in vivo. Taken together, our findings provide comprehensive preclinical evidence that n-3 PUFAs efficiently prevent ACC growth by inhibiting mTORC1/2, which may have important implications in the treatment of ACC.

Apoptosis of human prostate cancer cells induced by marine actinomycin X2 through the mTOR pathway compounded by MiRNA144.

The present study aimed to determine whether actinomycin X2 (AX2) intercepted the mTOR/PTEN/PI3K/Akt signaling pathway to inhibit human prostate cancer cells (PC-3) in vitro. The effects of AX2 on mTOR, PTEN, PI3K, and Akt at the protein level and mRNA were determined by western blotting and real-time reverse transcription-PCR (RT-PCR), respectively. Concurrently, the effects of AX2 on expression levels of MiRNA144 and MiRNA126 in PC-3 were measured by real-time RT-PCR. The association of MiRNA144 with 3'-UTR of mTOR was identified using the Dual-Luciferase Reporter Gene System. The direct effect of MIRNA144 on the mTOR/PTEN/PI3K/Akt pathway was determined by real-time RT-PCR and western blotting. Apoptosis of PC-3 cells induced by AX2 was determined by MTT and flow cytometry. The results indicated that mTOR/PTEN/PI3K/Akt were decreased and PTEN was increased by AX (1, 10 µmol/l) at protein and mRNA levels in a dose-dependent manner. MiRNA144 was decreased, whereas MiRNA126 was increased by AX2. MiRNA144 associated with 3'-UTR of mTOR was corroborated. Overexpression of MiRNA144 decreased mTOR, but did not affect PTEN, PI3K, or Akt. The proliferation rates of AX2 on PC-3 cells were decreased. It suggests that AX2 induces apoptosis of PC-3 cells via meddling in the mTOR/PTEN/PI3K/Akt signaling pathway, but those effects are compounded by MiRNA144. Both AX2 and MiRNA144 intercept the signaling in different ways but cross on mTOR.